Örebro University
Institution of Medical Sciences Bachelor thesis, 15 HP
January 2020
Writer: Anna Sundström
Mentor: Julia König & Julia Rode Nutrition-Gut-Brain Interactions Research Center (NGBI) Örebro University
The effect of faecal microbial
transplantation on mucosal CD8
+
T cells
in the colon of patients with collagen
colitis
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Abstract
Background. Our intestinal microbiota has many valuable functions. A defect response to –
or a dysbiosis of the microbiota has been associated with diseases such as microscopic colitis (MC), a condition with chronic diarrhoea. MC can be divided into the subtypes collagen colitis (CC) and lymphocytic colitis. MC is commonly treated with locally active
corticosteroids but relapse often occurs after completed periods of treatment. Faecal microbial transplantation (FMT) has recently been tested in one patient with CC and shown positive results in both symptoms and mucosal immune cell composition.
Aim. Our aim was to study the effect of FMT on mucosal CD8+ T cells in patients with CC.
Method. Immunohistochemistry was performed to visualize CD8+ cells in tissue samples from biopsies collected at baseline and six weeks after FMT. The difference in amount of cells was analysed using counting of cells and statistics.
Result. Ten CC patients participated. The mean number of cells in the tissue within an area of
0.200 mm² was 125.7 ± 43.1 before and 127.1 ± 48.7 after FMT. The mean difference was 1.4 ± 47.4. The change was not statistically significant (p = 0.93). On an individual level, both increases and decreases in the number of CD8+ cells after FMT were detected.
Conclusion. Many factors indicate FMT as a promising treatment for CC. While our result
showed no consistent change amongst the CD8+ cells other immune cells could be affected. Further studies should be executed to investigate the outcome of FMT in a larger group of patients.
Key words: Microscopic colitis, collagen colitis, microbiota, gut microbiota, faecal microbial
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Abbreviations
CC = Collagen colitis
C. difficile = Clostridioides difficile
FMT = Faecal microbial transplantation IBD = Inflammatory bowel disease IBS = Irritable bowel syndrome IEL = Intraepithelial lymphocytes LC = Lymphocytic colitis
LPL = Lamina propria lymphocytes MC = Microscopic colitis
NSAID = Nonsteroidal anti-inflammatory drugs PPI = proton pump inhibitors
SCFA = Short-chain fatty acid SD = Standard deviation TBS = Tris buffered saline UC = Ulcerative colitis
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Table of content
1 Background ... 4
1.1 Intestinal microbiota and its function ... 4
1.2 Gut microbiota in disease ... 5
1.3 Microscopic colitis ... 5
1.4 Faecal microbial transplantation ... 6
2 Aim... 7 3 Methods ... 7 3.1 Study design ... 7 3.2 Study population ... 8 3.3 Donors ... 8 3.4 Patients ... 8 3.5 Informed consent ... 10
3.6 Collection & preparation of faecal samples used for the FMT... 10
3.7 Faecal microbiota transplantation (FMT) procedure ... 10
3.8 Adverse events assessment... 11
3.9 Biopsies ... 11
3.10 Questionnaires, health forms and diary ... 11
3.11 Immunohistochemistry ... 11 3.12 Cell counting ... 12 3.13 Statistical analysis ... 12 3.14 Ethics ... 13 4 Results ... 13 4.1 Patient characteristics ... 13 4.2 Adverse events ... 14 4.3 Data ... 14 5 Discussion ... 15 5.1 Previous studies ... 16 5.2 Microbiota dysbiosis ... 17 5.3 FMT in other diseases ... 17 5.4 Limitations ... 18 6 Conclusion ... 19 7 Author’s contribution ... 20 8 Acknowledgements ... 20 9 References ... 21
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1 Background
1.1 Intestinal microbiota and its function
Humans are home to many bacteria and other microorganisms which are called the
microbiota. Microbiota can be found basically on every surface that is exposed to the external environment. Together we live in a coexistence. More than 70% of the microbiota are found in the gastrointestinal tract, probably due to its large surface area and its many molecules that can be used as nutrition for the bacteria [1]. The bacteria start colonizing us at birth. They can come from the mother when a baby comes through the birth canal and then from other people surrounding the baby in the beginning of its life. They are also determined by genetics [1] and by the food we eat [2].
Most of the bacteria in the gut microbiota are anaerobes of which the Bacteriodetes and Firmicutes are the two most common species [3]. Individuals have different compositions of bacteria in their gut but the core of the population is conserved and often shared by most humans [1].
The gut microbiota has a central role in the maturation of mucosal immunity in the intestines. The intestinal immune system includes Peyer’s patches, intestinal lymphoid tissues, immune cells and the epithelium itself [1]. The bacteria contribute to the immune system’s balance between homeostasis and inflammation [4]. The gut microbiota also helps to protect us from pathogens that reach the gastrointestinal tract by being the largest colony of bacteria. They do this by using many of the nutrients, production of antimicrobial substances and occupying possible attachment sites [1].
The bacteria contribute essentially to our metabolic processes that are needed for our
homeostasis [1]. The most extensively studied metabolites produced by the gut microbiota are short-chain fatty acids (SCFAs). SCFAs stimulate protection of the mucosa and the regulatory functions of the immune system. The SCFAs in stool from IBD patients is decreased and this is linked to a lower concentration of bacteria species producing butyrate as well as a low intake of dietary fibre [4]. Butyrate is an SCFA produced mainly from undigested dietary fibre by microbial fermentation by some of the bacteria in the gut microbiota, and it is very beneficial for good intestinal health. It is anti-inflammatory, strengthens the intestinal defence barrier and lowers oxidative stress, amongst others. One mechanism is that it inhibits NF-κB activation [5].
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Not having an excessive immune response against the microbiota of the gut is achieved through physical barriers between bacteria and host cells, modification of the antigens of the microbiota to make them less immunogenic or tolerance development in the immune cells [1]. The intestinal immune system shows a certain tolerance to the microbiota but also prevents it from overgrowth and infiltration by secreting IgA antibodies and antimicrobial peptides produced by immune cells in the mucosa [1].The majority of the differentiating T cells in a healthy intestinal mucosa become T-helper cells type 2 (Th2), which are a type of CD4+ T-cell, or regulatory T-cells which both promotes tolerance [1]. The intestinal mucosa also contains different types of CD8+ T cells. These are cytotoxic and target intracellular pathogens [6].
1.2 Gut microbiota in disease
In diseases such as IBD, which includes Crohn’s disease and ulcerative colitis (UC), one suggested pathophysiological mechanism is that the host immune system shows a defect response to the commensal gut microbiota [1]. An additional suggested pathogenesis involves a dysbiosis of the gut microbiota with a decreased diversity, increased groups of pathogenic bacteria and decreased groups of protective bacteria [4]. This is suggested to be the case in other diseases as well, even in some that are not directly connected to the intestines [1].
1.3 Microscopic colitis
Microscopic colitis (MC) was first described in 1985 [7]. During the 1980s and 1990s the incidence steadily increased but now seems to be stabilizing [8], with a reported prevalence of 219 [9] and 123 [10] per 100 000 inhabitants in 2010 [9] and 2008 [10], respectively. This was reported in the Western world. Bonderup et al. suggests that the increased incidence might be partly due to an increased diagnostic activity that results in increasing numbers of diagnosed patients [11]. The disease occurs more often in elderly women but is seen in both genders and all groups of age [8]. MC has been associated with several risk factors [7] such as intake of nonsteroidal anti-inflammatory drugs (NSAID) and proton pump inhibitors (PPI)[8], and smoking [12]. Onset of MC has also been seen after specific intestinal infections [8]. The diagnosis is based on clinical manifestation and macroscopic and microscopic
morphology of the colon [7]. The typical symptom is chronic non-bloody, watery, diarrhoea. The amount of stools differs from a few each day to over ten in one day. There are also more non-specific symptoms such as abdominal pain, fatigue, faecal incontinence, urgency and weight loss [7].
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The symptoms are the same for both lymphocytic colitis (LC) and collagen colitis (CC) which are subgroups of MC. However, the histologic morphology differs between the two
subgroups. The microscopic morphology of CC is intraepithelial lymphocytosis, increased cell infiltration into lamina propria, and a thickened subepithelial band of collagen that measures more than 10 µm [8]. Apart from the thickened collagen band the microscopic morphology is the same in LC but with a more abundant intraepithelial lymphocytosis [8]. The increased intraepithelial lymphocytes (IELs) in MC are CD8+ T-lymphocytes and they can cause epithelial damage [7]. The cells in the lamina propria are plasma cells, mast cells, neutrophils, eosinophils and lymphocytes [8] and they can induce inflammation [7].
Different theories exist regarding the pathogenesis of MC. One is that luminal agents give rise to an unrestrained mucosal immune response in predisposed individuals. These agents are not yet fully identified [8] but might be bacterial, dietary or some sort of medication that induce an inflammatory cascade in the mucosa [7]. Inflammatory processes may lead to reduced active absorption of sodium and chloride and reduced passive permeability through the mucosa, among other changes, which leads to a dysfunction of the collagen barrier and then diarrhoea [7].
The common treatment for MC is the locally active corticosteroid budesonide which results in improvement of clinical symptoms [8] and histology [7]. A disadvantage is that a relapse in the disease after completed periods of treatment is reported in many patients [13]. In the case of a mild disease anti-diarrhoeal medication can be used regularly or when needed [7]. Faecal microbial transfer has been tested as a non-pharmaceutical treatment option in one patient with MC (CC to be exact) in a case study performed in Örebro, Sweden [14].
1.4 Faecal microbial transplantation
In faecal microbial transplantation (FMT), stool from a healthy donor is administered to a patient that is assumed to have a gut microbiota dysbiosis. The new gut microbiota population is thought to restore the function of the intestine. The gut microbiota is normally assumed to be a part of or a cause of the conditions where FMT is used [15].
FMT is used as a second-line treatment for recurrent Clostridioides difficile (C. difficile) infections with efficacy tares of 90% and higher [15]. FMT is also being investigated as treatment for IBD [15], irritable bowel syndrome (IBS) [15,16], some autoimmune disorders, specific allergic diseases and metabolic disorders (obesity) [15]. In the case of IBD, FMT has shown positive results in patients with ulcerative colitis, but the efficacy is not as high as in
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treatment of C. difficile infections [15]. In one IBS study, FMT has given some short-term relief of symptoms [16]. The recommendations for FMT in IBD, IBS and metabolic syndrome need more evidence and therefore it should only be used as part of research when it comes to these conditions - not solely as a treatment [17].
The way to administer the FMT is chosen depending on the location of the disease and the capability of the physician executing it [17]. Colonoscopy is one of the most common ways to administer the faecal material as it has the greatest efficacy. The greatest risk is perforation of the intestine, especially in a compromised intestine. Sigmoidoscopy or enemas to the distal colon are used less frequently. Administration via the upper gastrointestinal or via capsules is also possible [15,17]. Risk during upper tract administration is vomiting and aspiration where the latter can lead to pneumonia and sepsis. Lower gastrointestinal tract administration is considered to be a safer option [17]. The donors of the faecal material are screened for various diseases that could be transmitted to the recipient via FMT [17].
In the case study mentioned earlier, FMT proved to have a beneficial effect on a CC patient. It resulted in less symptoms for 11 months after three treatments and a decrease in IEL and lamina propria lymphocytes (LPL) after only two treatments [14].
2 Aim
The aim was to study the effect of FMT on the number of mucosal CD8+ T cells in the colon of patients with CC, using immunohistochemistry where antigen-antibody interactions are utilised to locate specific antigens in tissues. This project was a part of a larger study of which the main purpose was to investigate if FMT could lead to improvement of symptoms in patients with CC.
3 Methods
3.1 Study design
The study was performed at Örebro University Hospital and Örebro University, Sweden, from April 2018 to October 2019. The immunohistochemistry analysis was performed from
November 2019 to December 2019. This thesis was a laboratory study and a part of a larger study in form of an intervention - and pilot study. Supplemental Figure 1 (Appendix 1) shows an overview of the study design of the larger study.
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3.2 Study population
The study included 10 patients with CC and two donors of faecal material.
3.3 Donors
Two healthy subjects who had a high proportion of butyrate producing bacteria in their faecal sample and were between 18 and 65 years of age were chosen as donors (out of 20-30
possible donors). They were found after looking into databases from earlier studies and after advertisements at Örebro University and Örebro newspapers. Exclusion criteria for donors are shown in Table 1. The donors were carefully screened regarding their medical background and went through blood and stool tests before being accepted as donors. They also went through these tests regularly during their participation in the study, and were asked about their health before every donation, to make sure they were always qualified as donors. The donors were asked to keep a stable diet during the study.
3.4 Patients
Ten patients, between 18 and 70 years of age, with a documented diagnosis of MC (subtype CC) who had an active disease participated in this study. The active disease was defined as more than three stools a day whereof at least one was watery and it was confirmed by interview. Their diagnosis was verified through biopsies (subepithelial collagen layer ≥ 10 µm) collected from the ascending colon during the first FMT. Exclusion criteria for participating patients are found in Table 1. During the study they agreed to stop their
budesonide treatment and to keep their diet stable. To monitor potential dietary changes they filled out a food frequency questionnaire before and after the study.
Patients were recruited within the region of Örebro. Patients with CC were informed about the study in conjunction with a routine visit to the gastroenterology’s endoscopy department at Örebro University Hospital or by sending information letters to patients with CC registered at the hospital. The patients who agreed to participate in the study were equally divided into two groups. Group 1 received FMT from donor A, and group 2 received FMT from donor B. The individual patients received faecal material from the same donor during the whole study.
Table 1: Exclusion criteria for FMT donors and Collagen colitis (CC) patients
Exclusion criteria for FMT donors Exclusion criteria for CC patients
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2. Known organic gastrointestinal disease 2. Recently diagnosed lactose intolerance (less than six months prior to first screening visit) 3. Recent (gastrointestinal) infection (within last six months) 3. Complicated gastrointestinal
surgery
4. First degree relative with Inflammatory bowel disease 4. Malignant disease except for non-melanoma skin cancer
5. First degree relative with cardiovascular thrombosis before 50 years of age
5. Major psychiatric disorder or other incapacity for adequate cooperation 6. History of or present gastrointestinal malignancy or polyposis 6. Severe depression
7. History of major gastrointestinal surgery 7. Dementia
8. Eosinophilic disorders of the gastrointestinal tract 8. Severe endometriosis 9. Human immunodeficiency viruses (HIV), hepatitis A, B, C or
known exposure within the recent 12 months
9. Recently diagnosed coeliac disease (less than six months prior to first screening visit)
10. Malignant disease and/or intake of systemic anti-neoplastic agents
10. Females who are pregnant or breast-feeding
11. Psychiatric disease or other incapacity for adequate cooperation 11. Regular intake of non-steroidal anti-inflammatory drugs (NSAIDs) 12. Chronic neurological/neurodegenerative diseases (e.g.
Parkinson’s disease, multiple sclerosis)
12. Antimicrobial treatment four weeks prior to first screening visit or antimicrobial prophylaxis
13. Metabolic syndrome 13. Abuse of alcohol or drugs
14. Obesity (BMI>30) 14. Regular consumption of probiotics
four weeks prior to randomisation 15. Autoimmune disease and/or patients receiving
immunosuppressive medications
15. Any clinically significant disease/condition which in the investigator’s opinion could interfere with the results of the trial 16. Major relevant allergies
17. Chronic pain syndrome 18. Chronic fatigue syndrome
19. Females who are pregnant or breast-feeding
20. Other chronic use of drugs that may affect the microbiome, e.g. proton pump inhibitors
21. Antimicrobial treatment or prophylaxis within the last three months
22. Abuse of alcohol or drugs
23. Tattoo or body piercing within the last 6 months 24. Participation in high-risk sexual behaviours
25. Travelling in countries with low hygiene or high infection risk for endemic diarrhoea within the last 6 months
26. Positive stool testing for Closteriodiales difficile, ova and parasites (e.g. Cyclospora, Isospora, Cryptosporidium), enteric
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pathogens (e.g. enterohaemorrhagic E. coli, Salmonella, Shigella, Yersinia, Campylobacter, Giarda antigen, amoebas) 27. Positive stool testing for multiresistant bacteria (e.g.
extended-spectrum betalactamase (ESBL) producing organisms, vancomycin-resistant enterococci (VRE) or methicillin-resistant Staphylococcus aureus (MRSA))
28. Calprotectin > 50 μg/g of faeces
29. Positive blood testing for HIV, hepatitis A, B, C, syphilis, human T-lymphotropic virus (HTLV), cytomegalovirus (CMV) and Epstein Barr virus (EBV)
30. Known clinically significant abnormal laboratory values 31. Any clinically significant disease/condition which in the
investigator’s opinion could interfere with the results of the trial
3.5 Informed consent
A written informed consent was signed by the donors and the patients before they could participate in the study. They were able to discontinue the study at any point of time without providing a reason.
3.6 Collection & preparation of faecal samples used for the FMT
The donors were asked to avoid eating nuts one week before the donations, in case of allergies in the patient group. Faecal material collected from the donors were covered with sterile saline (0.9% NaCl, 150 ml per 30g) and carefully stirred by hand to avoid oxygen exposure. The sample was amended with pharmaceutical grade glycerol to a concentration of 10% before being frozen at -80 °C. This process was performed within two hours after the
donation. The transplant was thawed at 37 °C directly before the FMT. Each preparation was used within three months for the transfers.
3.7 Faecal microbiota transplantation (FMT) procedure
The day before the first procedure the patients underwent a whole bowel cleansing, to clean out any residual faecal material, using the laxative picoprep. The first FMT was given via whole colonoscopy into the caecum and the amount of faecal material was 30 g in 150 mL. Midazolam and alfentanil were used for conscious sedation.
Before the procedure the patients were given 2 mg loperamide to prevent diarrhoea during and after the transfer. They were offered 2 mg more if needed after the procedure. After the biopsies for confirmation of the disease were obtained, the faecal suspension was
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Two and four weeks after the first FMT, two more FMTs were administered via enema and the amount of faecal material was 60 g in 300 mL each time. After receiving the enemas, the patients were kept in recumbent position for at least an hour in order to improve the
distribution of the faecal material. During this time, they were slowly moved from a left to a standard recumbent, and finally, right recumbent position. There was no bowel cleansing before the second and third FMT.
3.8 Adverse events assessment
The patients were asked to fill out a form about adverse events including body temperature after the procedure and to report any later adverse event to the study team.
3.9 Biopsies
Mucosal biopsies were collected from each patient by sigmoidoscopy from the mid-section of the descending colon at two different occasions: one week before the first FMT (baseline) and six weeks after. There was no whole bowel cleansing before this procedure to avoid removal of the newly introduced donor microbiota. These biopsies were used for different outcome analyses, while the biopsies collected at the right flexure during the colonoscopy for the first FMT were used for confirmation of the CC diagnosis.
The biopsies used for immunohistochemical analysis were formalin-fixed and embedded in paraffin according to routine at the Department of Pathology at Örebro University Hospital. They were then cut into 4 μm thick tissue samples that were mounted on glass slides.
3.10 Questionnaires, health forms and diary
As a part the larger study the patients completed different forms and questionnaires and also a bowel movement diary. These were used to evaluate the FMT as a treatment and to see if it can lead to remission of MC.
3.11 Immunohistochemistry
The tissue samples on glass slides were heated in an oven at 60 °C for 60 minutes to start deparaffinization. Antigen retrieval was performed in a pressure cooker (Bio care Medical, Pacheco, California, USA) with the pre-treatment reagent Borg Decloaker (pH 9, Bio care Medical, Pacheco, California, USA) at 110 °C for 10 min. The samples were then
deparaffinised in diluted Hot rinse (Bio care Medical, Pacheco, California, USA) that was pre-heated in the pressure cooker.
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The slides were incubated with anti-CD8 antibody (mouse monoclonal, clone 4B11, 1:50, Novocastra, Leica Biosystems Newcastle Ltd, Newcastle, UK) that was diluted with tris buffered saline (TBS) buffer. The tissue samples were incubated in Peroxidazed 1 (Bio care Medical, Pacheco, California, USA) to block endogenous peroxidase before the use of the detection system.
MACH 1 Universal HRP-polymer detection kit (Bio care Medical, Pacheco, California USA) for chromogen staining was used to prevent non-specific binding and to visualize the binding of the anti-CD8 antibodies. In between each incubation the tissue samples were rinsed in TBS buffer (Bio care Medical, Pacheco, California, USA).
The counterstaining was performed with haematoxylin.
Pertex mounting medium for light microscopy (HistoLab products AB, Västra Frölunda, Sweden) was used to mount the slides and then they were each covered with a 24 x 24 mm cover glass (VWR, Radnor, Pennsylvania, USA). The slides were left in a fume cupboard overnight and then stored at 2-8 °C until analysis using a light microscope.
The chromogen staining was performed in an immunohistochemistry laboratory at Örebro University, Sweden. The detailed protocol used is enclosed in Appendix 2. The two slides from one single patient were always stained in parallel. Skeletal muscle was used as negative control and healthy colon mucosa was used as positive control. Positive and negative control stainings are found in Figure 2 and 3 (Appendix 3).
3.12 Cell counting
The glass slides with tissue samples were scanned in 20-fold magnification before analysis using Panoramic 250 Flash II (3DHISTECH Ltd, Budapest, Hungary) and the compatible program Automatic Brightfield Scan (3DHISTECH Ltd, Budapest, Hungary). CD8+ cells were counted in the program CaseViewer (Version 2.1, 3DHISTECH Ltd, Budapest,
Hungary), and the plug in programme called ‘Marker counter’, in three randomized fields of vision per tissue sample that were approximately 0.200 mm² each. If a tissue sample was too small to fit three fields of vision that size, the fields were divided into smaller ones that were about the same area in total. The counting was performed manually by one investigator (AS).
3.13 Statistical analysis
The statistical analysis was performed with the software GraphPad Prism 8 (Version 8.3.0, GraphPad Software, San Diego, California USA) for Windows. Shapiro-Wilk test was used to
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test if the there was a normal distribution of values. Paired t test was used to compare the samples before the FMT with the samples after the FMT. p <0.05 was considered significant. Mean values are shown as mean ± SD.
3.14 Ethics
The study has been approved by the Central Ethical Review Board of Uppsala (registration number 2017/072) and was registered at clinicaltrials.gov (number NCT03275467) on
September 7, 2017. The study was conducted according to the principles of the Declaration of Helsinki and its revisions.
The risks during this study could be complications during or after the FMT. Perforation of the colon is the greatest risk during a colonoscopy but even a successful procedure can be
uncomfortable. FMT can also lead to diarrhoea afterwards which can cause discomfort. Disease carried by the donors could also be a risk to the patients if they are not screened properly before donating the faecal material and if they would pass on an infectious disease for example. Even if the side effects of FMT are most often mild and transient, the possible long-term risks of FMT have not yet been identified. All patients were carefully informed about these potential risks.
The potential benefit of this study is if FMT proves to be a successful non-pharmaceutical treatment for MC that is normally treated with chronic use of locally active corticosteroids. A placebo group was not used in this study as the group of patients was not that large since this study will contribute to the design of larger, placebo-controlled studies.
The patients’ tissue samples were marked with study numbers so that the individual patients could not be identified.
4 Results
4.1 Patient characteristics
Ten patients between the age of 44 and 70 were included in this study. The patients did not use budesonide during the course of the study, the last dose was administered at least 11 days before the baseline visit.
In nine of the ten patients, CC diagnosis was confirmed histologically when analysing the biopsies collected from the ascending colon (at the right flexure) during the first FMT by colonoscopy. One patient’s diagnosis could not be confirmed histologically but the result
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from this individual was still included in the analysis due to the fulfilled inclusion criteria (based on the symptoms described in the interview).
Chronic inflammatory or co-morbid autoimmune diseases were present in four out of the ten patients. One of the patients had never smoked, five of them had smoked earlier in life and four were currently smokers. There were no reported dietary changes. An overview of the patient outcomes is found in Table 2.
Table 2. Overview of the patient outcomes throughout the study
4.2 Adverse events
No major adverse events were reported.
4.3 Data
The amount of CD8+ cells in each patient before and after FMT assessed by
immunohistochemistry are shown in Figure 4. Two slides per patient were analysed (one before, one after FMT) resulting in a total of 20 slides, and no slides were excluded. The cell numbers were normally distributed (Shapiro-Wilk test).
The mean number of cells in an area of approximately 0.200 mm² was 125.7 ± 43.1 before and 127.1 ± 48.7 after FMT. The mean differences between the two groups were 1.4 ± 47.4.
* Clinical active disease was defined as more than three stools per day whereof at least one watery. ** Histopathological analysis of biopsies collected by whole colonoscopy. *** Clinical remission was defined as less than three stools per day whereof less than one watery. This information was collected from the large study of which this thesis is a part of, their results are not yet published. **** Assessed using immunohistochemical chromogen staining with anti-CD8 antibodies and manual point counting.
Patient Clinical active disease at baseline * Histological confirmation of CC diagnosis ** FMT donor Clinical remission 6 w after the first FMT ***
Histological decrease of mucosal CD8+
cells in colon 6 w after the first FMT ****
A Yes Yes A Yes Yes B Yes Yes A No Yes C Yes Yes A No No D Yes Yes B No Yes E Yes Yes B Yes No F No Yes A Yes No G No Yes B Yes Yes H No Yes B Yes No I No Yes B Yes Yes J No No A Yes No
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Paired t test showed no significant difference (p = 0.93) between the number of cells in the biopsies collected before the FMT compared to the biopsies collected after the FMT. A representative example of a colon biopsy with stained CD8+ cells before and after FMT is
shown in Figure 5. See Table 3 (appendix 4) for a complete presentation of the 20 stained tissue slides. Before FMT After FMT 0 50 100 150 200 250 C D 8 + c el ls
Figure 4. CD8+ cells present in microscopic colitis patients’ colon mucosa before and after faecal
microbial transplantation. The values are a mean of three areas of point counting per tissue sample. Circles represent participants with a decrease of CD8+ cells after FMT. Triangles represent
participants with an increase of CD8+ cells after FMT.
5 Discussion
The results above were achieved through immunohistochemical staining and analysis using a bright field microscope. The staining with anti-CD8-antibodies revealed a colonic mucosa
CD8+cells i colon mucosa. One field of vision visible. Before FMT - at baseline
CD8+cells in colon mucosa. One field of vision visible.
After FMT - at six weeks
Figure 5. Representative example of a colon biopsy with chromogen stained CD8+ cells. FMT = Faecal microbial transfer.
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with many CD8 antibody-antigen interactions in accordance with the histopathology of CC. The comparison between the biopsies collected at two different occasions (baseline and six weeks after FMT) showed the effect of FMT on the CD8+ cells in colon mucosa in patients
with CC, though they revealed no significant change in the number of CD8+ cells. According to our results, five patients had an increase and five patients had a decrease of CD8+ cells after FMT, with a rather wide range of values. There was a mean difference of 1.4 ± 47.4 between the two groups, in others words a general increase from the biopsies before to the biopsies after FMT. However, this change was not statistically significant.
5.1 Previous studies
To the best of our knowledge, this is only the second study investigating the change in CD8+
cells in CC after FMT. The result in our study is not in accordance with the previous case study performed on a CC patient by Günaltay et al [14]. They saw a general decrease in CD8+ lymphocytes in the colon mucosa after two FMTs. This study group used flow cytometry instead of immunohistochemistry as a method of quantification of cells and presented their result in percentage of the total number of T cells instead of mean number of cells [14]. This makes the two results difficult to compare. However, flow cytometry was used by the study team in the larger study that this thesis originates from, with the aim to see the change in IEL and LPLs in the CC patients after FMT. Their results are not yet published but worth
mentioning. They also did not see a significant change in CD8+ cells. Nonetheless, the
change in CD8+ T cells is in accordance with the result achieved by immunohistochemistry in this thesis and shows that both methods of analysis give similar outcomes.
Worth mentioning is that Günaltay et al. only included one patient [14] while we had a group of 10 patients. We had patients with a change in number of cells in certain directions after FMT but as a group the change was not unanimous.
Günaltay et al. also performed three FMTs on their patient but over a longer period of time, though their last biopsies were collected after the second FMT. Their second and third FMT were four and six months after the first one [14], while in our study all three FMTs were given within approximately four weeks. A longer period in between the treatments might be
beneficial for the long-term result, however, this has not been investigated yet. Günaltay et al. also had a different approach in terms of administration of the faecal material. Their first infusion was administered by enema and was given five days in a row according to their standard procedure for treating C. difficile, while the second and third infusion was by
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colonoscopy [14]. Trying other procedures of administration might be an idea for any future studies in the area.
In one of the studies performed in CC patients to evaluate the immune cell activity after budesonide treatment, the number of CD8+ cells was decreased to levels closer to those of healthy controls after treatment [18]. This study had a rather small number of participants but its results remain intriguing [18].
5.2 Microbiota dysbiosis
FMT is performed and tested as treatment in conditions related to microbial gut dysbiosis [15]. The mechanism of CC (and MC) in general have not yet been made clear but microbiota dysbiosis is considered one [14,19]. The result of Günaltay et al. support the hypothesis that factors in the lumen that could either be the microbiota, or affect the microbiota, might be involved in the pathogenesis of CC [14]. In a study performed by Rindom Kroogsgard et al. where a change in microbiota composition after budesonide treatment was investigated, a lower diversity in both LC and CC patients before the treatment compared to healthy controls was found. After the budesonide treatment the diversity was more similar to that of the controls [20]. According to Millien et al., pro-inflammatory bacteria families associated with PPI and NSAID intake are increased in patients with MC [22]. This provides a rationale for FMT and the ability to treat a potential unbalanced microbiota in MC patients.
One thing to remember is that dysbiosis has been considered both a cause and a consequence to intestinal inflammation in IBD [4]. In MC the full pathogenesis is still unspecified but is assumed to be multifactorial [8] and there are several theories to the main symptom diarrhoea [7]. If the dysbiosis would only be a consequence, it is questionable if a new microbiota could result in remission of the disease.
5.3 FMT in other diseases
Onset of MC has been seen after intestinal infections with Yersinia enterocolitica,
Campylobacter jejuni and C. difficile [8]. C. difficile infection is the result of an overgrowth
of this pathogen in the intestine and is often caused by dysbiosis due to antibiotic use [1]. FMT is used as a second-line treatment with success rates of 92% in recurrent C. difficile infections [15]. Bruno et al. showed preliminary results where a decrease in activated CD8+ T-cells in C. difficile infected patients treated with FMT occurred [23] which shows that FMT can affect CD8+ cells.
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There is increasing evidence that lower diversity and a decreased stability in the gut
microbiota is present in patients with UC [17]. Carstens et al. [19] compared the gut dysbiosis in IBD (including UC) to that in MC. Their results suggested that the diseases both might have mechanisms related to the microbiota as part of their pathogenesis [19]. FMT has shown promising result on UC as well [24] and due to the similarities with MC, this also promotes FMT as a promising treatment for MC.
5.4 Limitations
In immunohistochemical stainings performed in a study by Göranzon et al., a general increase of IEL and LPL was shown in MC compared to healthy controls, whereof the CD8+ cells were
increased in both IEL and LPL and the CD4+ cells were decreased in the IEL [25]. The
original plan for the lab work in this thesis was to perform a double-staining for both CD8+ and CD4+ cells, but the protocol showed no good result with the CD4-antibody, and the establishment of a new protocol was out of the scope of this thesis. A successful double-staining would have given more information about the normalization of the patients’ mucosa after FMT.
An additional aspect to consider is that the increase in intraepithelial lymphocytes is more distinct in LC patients than CC patients [25] and that raises the question if patients with the subtype LC could have been a better group to use in this study. In LC there are more than 20 intraepithelial lymphocytes per 100 surface epithelial cells [25] and this could have been a good starting-point while analysing the biopsies taken after the FMT. However, there was another analysis that was supposed to be performed in this project that could not have been performed in LC patients and that was an evaluation of the mucosal collagen layer. This could not be carried out due to the absence of an analysable collagen layer in some of the tissue samples. The absence of an incomplete collagen layer is hard to prevent since it cannot be controlled how much of the tissue layers stays intact while collecting biopsies. The biopsies that were analysed were collected from the descending colon and the biopsies used for histological confirmation of a diagnosis were collected from the ascending colon. This has probably also affected our ability to analyse the collagen layer since the thickened collagen layer in CC patients is most distinct in the more proximal colon (ascending and transverse parts) and can be absent in the more distal parts [8].
The statistical results of this thesis could have been affected by the fields of vision only being three in number. In the immunohistochemical investigation of immune cells in MC performed
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by Göranzon et al. [25], ten fields of vision were used on every slide and if fewer than four could be measured the slide was excluded. The area of their fields of vision were also larger [25]. Six fields of vision, or more, would have been preferable in this thesis but could not be achieved in each biopsy due to the small size of some of them. A mean number from more fields of vision per tissue sample could have had an effect on the result and provided a mean number closer to the true amount.
The number of patients participating is also a factor affecting the result. With a larger group of patients, we could have seen a reaction to FMT closer to that of the general population of CC patients and maybe presented a more trustworthy result. However, this was a pilot study only.
In addition, it could be that any short-term change in immune cells in the intestinal mucosa might also be a reaction to the introduction of new and unknown luminal antigens. Antigens related to medication, diet or bacteria were suggested to cause an inflammatory cascade via CD8+ cell activation in MC [7]. Possibly, the changes observed in the biopsies six weeks after baseline were still a reaction to the new microbiota. It would therefore have been interesting to collect biopsies at a third occasion scheduled a few months after the first FMT, when the intestinal immune system has reached homeostasis. As mentioned earlier, the time from baseline to the last biopsy was not very long.
The choice of donors might also affect the result in studies using FMT. In a study on FMT in UC patients, the most successful FMTs came from two of the four donors [24]. While this study investigated the ability of FMT to induce remission [24], a so-called donor effect might also be applicable to changes in CD8+ cells in the mucosa. However, we did not see any connection of that sort.
6 Conclusion
During this thesis the short-term (six weeks) effects in terms of changes in CD8+ cell number after FMT were recorded. Due to the small number of participants in this study it can be difficult to achieve evident and significant results. The change between the number of cells at baseline and six weeks after FMT was in an upwards direction but not significant. Many possible factors might be influencing this outcome, as discussed above, and maybe the cells affected by FMT were not the CD8+ after all. However, this was only a pilot study and FMT should be further investigated as a non-medical treatment in CC patients using a larger group of patients.
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7 Author’s contribution
The author contributed to the study by performing the immunohistochemical staining, scanning of the stained slides with tissue samples, manual counting of cells and statistical analysis.
8 Acknowledgements
I would like send a great thank you to my mentors Julia König and Julia Rode who have guided me through the process of my bachelor’s thesis. With your guidance I have gained a lot of knowledge and an interest in future research. I also want to say thank you to Anna Göthlin whom I have been able to ask about the lab – and analysing equipment during my days in the laboratory.
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Appendix 1
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Appendix 2
21/11 2019
Protocol, single stain
Anti CD8 antibody + DAB detection system
1. Heat slide in oven at 60 °C for 60 min. 2. Deparaffinization & antigen retrieval (AR)
With hot rinse and Borg (pH 9) & pressure cooker.
Fill the pressure cooker with 500 ml deionized water. Place three containers in a holder. Fill one with Borg, one with hot rinse (1 ml per 24 ml deionized water) and one with deionized water.
Put the slides in the container with Borg & start the pressure cooker.
Heat in pressure cooker at 110 °C for 10 min. Open pressure cooker at 95 °C, take out slides at 70 °C.
Dip in hot rinse 20 times and incubate 1 min. Rinse in TBS 2 min. Change the TBS.
Make a circle around the tissue with the hydrophobic pen, >3 mm margins. 3. Protein block
Put two drops of Bio care’s Background sniper on each slide. Incubate for 15 min at room temperature (RT).
Rinse in TBS 2 min.
4. Peroxide block (OBS sensitive of light)
Use a pipette and drop 50 µl of Bio care´s Peroxidase 1. Incubate 5 min at RT.
Rinse in TBS 2 min.
Use the hydrophobic pen again if necessary. 5. Primary antibody
Mix antibody and TBS with 1:50 dilution. Make enough to cover each slide with 50 µl. Put a wet paper in the incubation box, this can remain during all incubations.
Incubate in antibody solution for 30 min at RT. Rinse in TBS 2 min.
6. Probe
Put two drops of MACH 1 mouse probe on each slide. Incubate for 15 min at RT.
25 7. HRP-Polymer
Put two drops of HRP-Polymer on each slide. Incubate for 30 min at RT.
Rinse in TBS 2 min. 8. Chromogen
Mix 8 µl DAB chromogen per 250 µl DAB substrate buffer. Make enough to cover each slide with 50 µl.
Incubate for 5 min at RT.
Drip DAB off on a paper. Rinse in TBS 2 min. All DAB waste goes in a special container!
Put the slide in a rack in a container filled with deionized water. 9. Counterstain
Incubate in haematoxylin for 1 min.
Rinse in container with deionized water. Put container with slide under the tap with normal water for 1 min.
Dry in oven at 60 °C.
10. Mount slides using mounting medium and cover it with a cover glass. Let dry for at least 30-60 min.
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Appendix 3
Figure 2. Healthy colon mucosa, positive control to chromogen staining of CD8+ cells.
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Appendix 4
Table 3. CD8+ cells in colon mucosa of patient with Microscopic colitis before and after FMT*
Patient CD8+ cells before FMT - at baseline CD8+ cells after FMT - at six weeks
A B C D E F G H I J
